HDAC抑制剂对脉络膜黑色素瘤细胞系C918细胞增殖的影响及相关机制

目的：阐明组蛋白去乙酰化酶(HDAC)抑制剂辛二酰苯胺异羟肟酸(SAHA)对脉络膜黑色素瘤(CM)细胞系C918细胞增殖的影响并探讨相关机制。

方法：使用倒置荧光显微镜观察不同浓度SAHA(0.625、1.25、2.5μmol/L)对C918细胞形态的影响; CCK-8法观察C918细胞活力的变化; 细胞平板克隆形成实验和EdU染色法观察SAHA对C918细胞增殖的影响; 同时,Western blot检测细胞增殖相关蛋白c-Myc、细胞周期蛋白CyclinA2和CDK2以及HDAC7和成纤维细胞生长因子18(FGF18)的表达。

结果：与空白对照组比较,光镜下见SAHA可减小C918的细胞密度,促进细胞皱缩,且随着SAHA浓度的增加对细胞的抑制作用也增强; CCK-8法检测结果显示SAHA浓度依赖性抑制C918细胞活力,2.5μmol/L浓度时抑制率达80%; Western blot结果表明SAHA可呈浓度依赖地降低C918细胞中的增殖蛋白c-Myc、细胞周期蛋白CyclinA2和CDK2的表达; 另外,1.25μmol/L SAHA显著降低EdU染色阳性细胞数和细胞克隆数。更为重要的是,与空白对照组相比,SAHA能浓度依赖地降低HDAC7和FGF18的表达。

结论：SAHA能够通过抑制HDAC7/FGF18信号通路抑制CM细胞系C918细胞的增殖。     

曲古抑菌素A抑制TGF-β1诱导人Tenon囊成纤维细胞活化的作用

目的：探讨组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitor,HDACi)曲古抑菌素A(trichostatin A,TSA)对TGF-β₁诱导人Tenon囊成纤维细胞(human Tenon fibroblasts, HTFs)增殖及Ⅰ型胶原纤维合成的影响。

方法：首先,将不同浓度(200、400、600、800nmol/L)TSA作用HTFs共24h后,MTT法检测其对细胞活力的影响; 然后,不同浓度(400、600nmol/L)TSA与5ng/mL TGF-β₁混合作用于HTFs共24h,MTT法检测其对细胞活力的影响; 最后,RT-PCR和Western-blot法分别检测不同浓度(400、600nmol/L)TSA与5ng/mL TGF-β₁混合以及600nmol/L TSA对HTFsⅠ型胶原纤维的mRNA及蛋白表达的影响。

结果：MTT证实,与对照组相比,400nmol/L及以上浓度TSA作用组,HTFs活力显著下降(P<0.05); 两种浓度(400、600nmol/L)TSA均可减弱TGF-β₁促HTFs增殖的作用(P<0.05); RT-PCR和Western-blot证实两种浓度(400、600nmol/L)TSA对TGF-β₁诱导上调的Ⅰ型胶原纤维在基因转录及蛋白表达水平有逆转作用。

结论：TSA能够抑制TGF-β₁诱导的HTFs增殖,并减弱Ⅰ型胶原纤维基因转录及蛋白表达水平。     

曲古抑菌素A对兔眼滤过术后滤过泡作用的研究

目的：观察曲古抑菌素A( trichostatin A,TSA)作用于兔眼滤过术后滤过泡的形态变化,研究其对术后结膜瘢痕的抑制作用。
　　方法：兔眼滤过术中结膜下注射TSA、丝裂霉素C( MMC)、PBS,分别于术后3,7,14,21,28d应用Krofeld评分评价滤过泡的形态变化。
　　结果：TSA组14 d内滤过泡弥漫性隆起,28 d囊性泡形成。术后14,21 d TSA组滤过泡评分高于PBS组,具有统计学差异(P<0.05)。
　　结论：TSA能够抑制术后结膜瘢痕形成,延长滤过泡存在时间,保持滤过道通畅。     

表基因修饰参与H2O2介导人晶状体上皮细胞SRA01/04的生长停滞

目的 探讨在H2O2介导人晶状体上皮细胞SRA01/04生长停滞下,表基因修饰的改变.方法 将培养的人晶状体上皮细胞SRA01/04分为3组,分别为:A组:先加H2O2 组,即在细胞培养液内先加0.5 μmol·L-1 H2O2,30 min后再加吡诺克辛液300uL培养23.5 h;B组:后加H2O2组,印先在细胞培养液内加吡诺克辛钠液300μL培养23.5 h后,再加0.5μmol·L-1 H2O2培养30 min;C组:对照组,即不加H2O2及药物培养24h.对各组细胞进行免疫细胞化学检测和Western bolt检测,分析各组细胞内组蛋白脱乙酰化酶(histone deacetylase, HDAC)1、核因子-κB(nuclear factor-Kappa B,NF-κB)、c-myc及周期蛋白1(Cyclin D1)的表达.结果 免疫细胞化学检测结果显示,NF-κB表达信号的灰度值:A组(192.25±5.56)＞B组(170.40±2.50) ＞C组(157.14±1.09),3组间两两相比差异均有统计学意义(均为P ＜0.01);HDAC1、c-myc及Cyclin D1表达信号的灰度值均为C组＞B组＞A组,且3组间两两相比差异均有统计学意义(均为P＜0.01).Western blot检测结果显示,相较于对照组,以H2O2处理人晶状体上皮细胞SRA01/04后HDAC1、Cyclin表达减弱,并且A组比B组更为明显;同时可见NF-κB的表达增强,A组也比B组更为明显.结论 在H2O2介导人晶状体上皮细胞SRA01/04生长停滞下,HDAC1活性减弱,NF-κB激活,c-myc及Cyclin D1的表达下调.     

人Tenon囊成纤维细胞的体外培养及增殖能力研究

目的:离体培养人Tenon囊成纤维细胞(HTF)并观察细胞增殖能力。方法:取斜视患者手术切除Tenon囊组织进行成纤维细胞原代培养,培养的细胞通过免疫荧光染色法进行鉴定。CCK-8法描述细胞生长曲线,测定细胞活力。流式细胞术分析细胞周期,计算增殖指数。结果:通过组织块法成功培养出HTF。不同代次原代培养的HTF之间增殖能力无明显统计学差异(P>0.05)。结论:HTF在体外易于培养,经过数次传代,增殖能力稳定,是进行Tenon囊抗纤维化研究的良好靶细胞。     

TGF-β1对Tenon囊成纤维细胞增生和HSP47表达的影响

目的探讨转化生长因子B1（TGF-β1）对人Tenon囊成纤维细胞（HTF）增生和热休克蛋白47（HSP47）表达的影响。方法青光眼患者Tenon囊组织体外培养，用不同质量浓度0．01、0．1、1、5、10ng／mLTGF-β1处理细胞，继续培养24h，用MTT比色法检测吸光度值（A），用免疫细胞化学检测HSP47和Ⅰ型胶原纤维蛋白含量，用实时荧光定量PCR检测HSP47和Ⅰ型胶原mRNA表达。结果TGF-β1能促进HTF的增生（P〈0．05），诱导HTF表达HSP47和Ⅰ型胶原纤维蛋白（P〈0．05），促进HTF表达HSP47和Ⅰ型胶原纤维mRNA（P〈0．05），其作用随TGF-β1质量浓度的增加而增强。结论TGF-β1通过促进HTF纤维增生，诱导下游介质HSP47和Ⅰ型胶原蛋白表达而促进HTF纤维化，可能是青光眼滤过手术后瘢痕形成的重要机制。     

缓激肽刺激体外培养的视网膜色素上皮细胞炎症反应的作用机制

目的：探讨缓激肽刺激体外培养的视网膜色素上皮（ retinal pigment epithelium , RPE ）细胞炎症反应的作用机制。方法：体外培养 ARPE－19细胞,100 nmol／L 缓激肽（bradykinin,BK）刺激24h后,光镜下观察细胞形态变化,细胞免疫荧光检测BK受体定位;共聚焦显微镜检测BK及其拮抗剂作用下Ca2＋变化;Western blot 检测对照组与100 nmol／L BK 处理24 h 后（ BK 组） COX－1、COX－2、eNOS、iNOS蛋白的表达量。结果：BK刺激后,ARPE－19细胞形态无明显变化;ARPE－19细胞可检测到激肽B1、B2受体;与对照组相比, BK组Ca2＋浓度显著升高;B1R拮抗组及B2R拮抗组的Ca2＋浓度较BK组升高幅度降低,B1R及B2R拮抗组Ca2＋浓度较对照组无明显变化;BK作用ARPE－19细胞后, COX－2及iNOS蛋白含量显著增加（P＜0．001）。结论：BK通过与B1 R及B2 R结合促进体外培养的ARPE细胞COX－2及iNOS表达增加。     

曲尼斯特对人眼Tenon囊成纤维细胞TGF-β1，2 mRNA表达的影响

目的观察曲尼斯特对人眼Tenon囊成纤维细胞(human tenon fibroblast,HTF)TGF-β1、TGF-β2 mRNA表达的影响.方法体外进行HTF细胞原代及传代培养.采用半定量逆转录聚合酶链反应(RT-PCR),研究不同浓度和不同作用时间的曲尼斯特对HTF细胞TGF-β1、TGF-β2 mRNA表达的影响.结果曲尼斯特作用24 h后,25.0、50.0、100.0 mg/L浓度曲尼斯特组与对照组相比均能够明显下调HTF细胞TGF-β1(P＜0.05、0.05、0.01)和TGF-β2(P＜0.01、0.05、0.01)mRNA表达.50.0 mg/L曲尼斯特处理12 h、24 h、48 h组相对于相应时间对照组,能够明显下调HTF细胞TGF-β1(P＜0.05、0.01、0.01)和TGF-β1(P＜0.01、0.01、0.01)mRNA表达.结论 曲尼斯特能够下调体外培养HTF细胞TGF-β1、TGF-β2 mRNA的表达,下调效应随药物浓度和作用时间的延长而加强.(中国眼耳鼻喉科杂志,2006,681～83)     

褪黑素对高糖刺激人视网膜色素上皮细胞诱导型一氧化氮合酶表达的影响

目的研究褪黑素对高糖刺激体外培养的人视网膜色素上皮（retinal pigment epithelial,RPE）细胞诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）表达的影响。方法培养人RPE细胞,分为对照组、甘露醇组、高糖组和高糖＋褪黑素组,作用48h后,光镜观察细胞形态;MTT法检测细胞活性;免疫荧光染色法和Western blot检测RPE细胞中iNOS的蛋白表达。结果MTT检测结果表明高糖可以抑制RPE细胞增生,加入褪黑素后,抑制作用减弱;免疫荧光染色法和Western blot检测结果表明,与对照组相比,高糖组iNOS蛋白表达明显增高,加入褪黑素后,iNOS表达被显著抑制。结论高糖可以抑制RPE细胞增生,诱导RPE细胞iNOS的表达上调,而褪黑素可减轻细胞损伤。     

人眼Tenon's囊成纤维细胞原代培养的研究

目的探讨体外培养原代人眼Tenon’s囊成纤维细胞的方法及其生长特性,为抗纤维化研究提供靶细胞模型。方法取翼状胬肉及斜视手术患者的Tenon’s囊组织,用组织块法培养原代成纤维细胞。用免疫荧光方法鉴定细胞。对细胞进行传代、冻存和复苏的观察。对复苏后的细胞行MTT法检测其活力并绘制生长曲线。结果人眼Tenon’s囊成纤维细胞可以在体外用组织块方法培养出来,呈典型的长梭形。免疫荧光鉴定细胞波形蛋白染色阳性。细胞多次传代后依然生长迅速,3 d即可长满瓶底。复苏后细胞2~6 d处生长对数期,增殖能力良好。结论人眼Tenon’s囊成纤维细胞体外生长状态良好,可以液氮冻存,复苏后细胞活力较强,可以用于抗纤维化增生的基础研究。     

Effects of varying 5-fluorouracil exposure duration on Tenon's capsule fibroblasts

Purpose : 5‐Fluorouracil (5‐FU) is used intraoperatively to improve the success of trabeculectomy. Common practice is a 5‐min application of 5‐FU delivered via a Weck Cel sponge. The aim of this study was to determine if shorter exposure times were as effective in inhibiting Tenon’s capsule fibroblast proliferation. Methods : Samples of Tenon’s capsules, obtained from young patients undergoing strabismus surgery, were immersed in vitro in 5‐FU (50 mg/mL) for periods ranging from 1 to 10 min. Control samples were exposed either to growth medium (M199) or 5‐FU vehicle alone. Explants were cultured for up to 7 days and analysed for cell density, cell viability using fluoroprobe detection, and cell proliferation determined by proliferating cell nuclear antigen (PCNA)‐staining. Results : Exposure to 5‐FU for 1 and 5 min resulted in a similar and significant inhibition of the culture‐induced increase in stromal cell density (P < 0.01), a significant reduction in the percentage of viable cells (P < 0.02), and reduced PCNA indices, compared with controls. Exposure times of 3 and 10 min produced similar results. Conclusions : In vitro, under conditions of organ culture, a 1‐min exposure to 5‐FU had a significant antiproliferative effect on fibroblasts of Tenon’s capsule, and was similar to 5 min of exposure. Optimal intraoperative 5‐FU exposure time may be as little as 1 minute.     

Antiproliferative effect of mycophenolate mofetil on cultured human Tenon fibroblasts

BACKGROUND:Wound healing after glaucoma filtering surgery is often complicated by exaggerated scarring of the subconjunctival Tenon's layer. Therefore, antiproliferatives are commonly employed. The immunosuppressive drug mycophenolate mofetil (MMF) is used to prevent graft rejection after kidney or liver transplantation. The effect is mediated by inhibition of lymphocyte proliferation. In this study we investigated the effect of MMF on human Tenon fibroblast proliferation in cell culture.METHODS:Human Tenon fibroblasts (HTF) were cultivated with 10% fetal calf serum. Cells were incubated with MMF concentrations of 0.1 microM to 3000 microM for up to 20 days. In a second set of experiments HTF were incubated for 10 min only in MMF solutions. Cell counts were performed to evaluate the proliferation rate. The proliferation was also assessed by Ki67 staining. Morphological changes were documented by vimentin staining.RESULTS:Growth inhibition of HTF by MMF was concentration dependent. IC(50) was 0.85+/-0.05 microM for 6 days of incubation. Brief exposure to MMF leads to a reversible growth arrest for up to 14 days with concentrations of 1000 microM or higher. Ki67 staining confirmed the concentration dependent proliferation rate.CONCLUSION:MMF has a concentration-dependent antiproliferative effect on HTF without any detected cytotoxicity in the applied concentration range. Brief incubation also leads to a growth arrest; therefore, intraoperative MMF application might prevent exaggerated scarring after glaucoma filtering surgery.     

Alkylphosphocholines: a new therapeutic option in glaucoma filtration surgery

PURPOSE: To investigate the effect of alkylphosphocholines (APCs) on human Tenon fibroblast (HTF) proliferation, migration, and cell-mediated collagen gel contraction. METHODS: HTFs were isolated from tissue samples of three patients obtained during surgery and cultured in DMEM and 10% fetal calf serum (FCS). HTFs (passage 3-6) were treated with one APC in different concentrations spanning the 50% inhibitory concentration (IC(50)), as determined previously. Inhibition of cell proliferation was assessed by the tetrazolium dye reduction assay. Migration was determined in chemoattractant chambers with fibronectin-coated polycarbonated membranes. For inhibition of contraction, three-dimensional collagen gels were seeded with HTFs, and the gel size was measured. Cell viability was determined by the trypan blue exclusion assay. For analysis of the mechanism of action, protein kinase C (PKC) activity was measured. RESULTS: The IC(50) varied between 7.0 and 10.5 microM for all APCs tested. At this concentration, all four APCs inhibited HTF migration and cell-mediated collagen gel contraction in the presence of serum. The inhibitory effects on HTF proliferation, migration, and contraction were observed at nontoxic concentrations. PKC activity was reduced to 50% of control level at the IC(50) of all APCs applied. CONCLUSIONS: APCs are effective inhibitors of HTF proliferation, migration, and cell-mediated contraction of collagen gels at nontoxic concentrations. Their mechanism of action seems to involve the inhibition of the PKC pathway.     

体外转染P27抑癌基因抑制人眼球筋膜囊成纤维细胞增生的研究

目的探讨阳离子脂质体介导的P27抑癌基因对体外培养的人眼球筋膜囊成纤维细胞增生的抑制作用。方法收集斜视患者矫正手术过程中获取的球筋膜囊组织制备成组织块,采用贴壁法在DMEM＋质量分数10%胎牛血清培养液中进行培养和传代。采用脂质体介导的方法,将构建的增强型绿色荧光蛋白（pEGFP）-N1P27真核表达质粒转染体外培养的人眼球筋膜囊成纤维细胞为pEGFP-N1P27转染组,同时以重组质粒pEGFP-N1的空质粒转染组为空载体组,另设部分未转染的成纤维细胞为未转染组。Westernblot法检测P27蛋白在人眼球筋膜囊成纤维细胞中的表达,用流式细胞技术进行细胞周期分析,MTT法检测P27基因对培养的人眼球筋膜囊成纤维细胞活性的影响,检测值以细胞的吸光度（A490）值表示。结果荧光倒置显微镜下见转染成功的成纤维细胞呈绿色荧光。Westernblot检测显示,pEGFP-N1P27转染组中可见宽大的蛋白条带,证实人眼球筋膜囊成纤维细胞在蛋白水平表达P27基因。细胞周期分析结果表明,pEGFP-N1P27转染组73.16%的人眼球筋膜囊成纤维细胞处于G0-G1期,26.84%处于S期,而空载体组的人眼球筋膜囊成纤维细胞在G0-G1期、S期者分别为63.29%和58.16%,未转染组分别为36.71%和41.84%。MTT法检测表明,pEGFP-N1P27转染组人眼球筋膜囊成纤维细胞的A490值为0.079±0.054,明显低于空载体组的0.127±0.106和未转染组的0.180±0.007,差异均有统计学意义（P=0.000,P=0.011）。结论转染的P27基因对体外培养的人眼球筋膜囊成纤维细胞的增生具有抑制作用,并能够抑制成纤维细胞的活性。     

小檗胺抑制兔Tenon囊和滤过道内成纤维细胞增殖的实验研究

目的:研究钙调素拮抗剂小檗胺(berbamine,BER)对兔Tenon囊成纤维细胞和兔小梁切除术后滤过道内成纤维细胞增殖的抑制作用。方法:体外培养兔Tenon囊成纤维细胞,经不同浓度BER处理不同时间后,细胞计数Kit8(CCK8)法检测BER对兔Tenon囊成纤维细胞增殖的抑制作用,流式细胞仪检测其凋亡率及细胞周期的变化。HE染色检测BER对兔小梁切除术后滤过道内成纤维细胞增殖的抑制作用。结果:兔Tenon囊成纤维细胞经不同浓度BER处理不同时间后细胞增殖受到抑制,并呈时间剂量依赖性。流式细胞仪检测发现BER处理后兔Tenon囊成纤维细胞呈现G1期阻滞,细胞凋亡率明显增加,20mg/LBER处理9h,细胞凋亡率从0.64%增加到31.86%。HE染色显示BER显著抑制兔小梁切除术后滤过道内成纤维细胞的增殖。结论:小檗胺在体内外均能抑制成纤维细胞的增殖,其可能是通过诱导细胞凋亡方式抑制兔Tenon囊和滤过道内成纤维细胞的增殖。     

The effect of ICG on mitomycin C cytotoxicity in human tenon fibroblasts

PURPOSE: To examine the effects of indocyanine green (ICG) with and without mitomycin C (MMC) on proliferation of cultured human Tenon fibroblasts. METHOD: Fibroblast monolayers were exposed to either MMC [0.4 mg/mL in phosphate buffered saline (PBS)] or PBS containing ICG (0.0625%, 0.125%, 0.25%, and 0.5% in 200 microL PBS) or a combination of MMC (0.4 mg/mL in PBS) and ICG (0.25% and 0.5%) for 5 minutes. Controls were exposed for 5 minutes to MMC, PBS, or culture medium containing no ICG. After treatment, the monolayers were washed and incubated in culture medium for 24, 48, 72 hours, and 1 week periods after which the number of viable cells was quantified. RESULTS: The presence of ICG alone, at concentrations ranging from 0.0625% to 0.5%, had no effect on the rate of fibroblast proliferation measured at any of the incubation periods. As expected, MMC treatment resulted in a significant reduction in viable fibroblast number (8.4+/-0.13x10(3)). ICG in combination with MMC did not significantly alter fibroblast numbers (8.5+/-0.05x10(3)) up to 1 week compared with MMC alone (8.4+/-0.12x10(3)). CONCLUSIONS: ICG at concentrations of 0.5% and below do not reduce proliferation of Tenon capsule fibroblasts. ICG did not potentiate or diminish the effect of MMC on Tenon capsule fibroblast proliferation.     

自体眼球筋膜囊充填修补术在外伤性角膜缺损手术中的应用分析

目的：调查研究自体眼球筋膜囊充填修补术在外伤性角膜缺损手术中的应用效果。
　　方法：采用随机数字表法将100例外伤性角膜缺损患者分成治疗组和对照组各50例,给予治疗组患者自体眼球筋膜囊充填修补手术治疗,给予对照组患者常规性的手术治疗。
　　结果：治疗组患者的临床治疗总有效率为88%,对照组患者的临床治疗总有效率为68%,两者相比具有显著性的差异(P<0.05),所有患者接受治疗过程中均未出现异常现象。
　　结论：采用自体眼球筋膜囊充填修补手术治疗外伤性角膜缺损,有较好的临床治疗效果,且手术安全性较高,值得在临床上进一步推广运用。     

阿昔洛韦对体外培养人眼Tenon囊成纤维细胞增殖迁移的影响

目的:探索阿昔洛韦(ACV)对体外培养人眼Tenon囊成纤维细胞(HTFs)的增殖和凋亡的影响以及作用机制。方法:将HTFs分为ACV处理组和空白组;CCK8检测不同浓度梯度下的细胞增殖速率,划痕实验检测HTFs迁移能力,流式细胞术检测HTFs凋亡以及细胞周期。结果:与空白组相比,ACV处理组(终浓度分别为1.125、2.25、3.375、4.5mmol/L)HTFs增殖速率显著降低(P<0.05),且呈浓度依赖性。4.5mmol/L ACV处理组划痕细胞迁移率显著降低(P<0.05),细胞凋亡率显著增加(P=0.0005),细胞周期G0/G1期峰值升高(P=0.0011),S期下降(P=0.0006),细胞周期被阻滞在G0/G1期。结论:阿昔洛韦可以通过阻滞HTFs周期促进细胞凋亡,抑制HTFs的增殖和迁移。     

光动力疗法抑制体外培养的人球筋膜囊成纤维细胞增殖的研究

目的　探讨光动力疗法 (photodynamictherapy ,PDT)对体外培养的人球筋膜囊成纤维细胞增殖的抑制作用。方法　将体外培养的人球筋膜囊成纤维细胞分成A至J组 ,每组 4孔。其中A至G组为PDT组 ,依次加入光敏剂苯并卟啉衍生物单环酸A ,使其终浓度分别为 2 5 0× 10 3g/L、1 2 5× 10 3g/L、0 6 2× 10 3g/L、0 31× 10 3g/L、0 16× 10 3g/L、0 0 8× 10 3g/L及 0 0 4× 10 3g/L ,15min后用波长为 6 89nm ,能量密度为 2 40J/cm2 的半导体激光照射。H组为丝裂霉素C(mitomycinC ,MMC)对照组 ,加入MMC使其终浓度为 0 2 0g/L。I组为空白对照组 ,未加任何处理。J组为单纯激光对照组。各组培养 2 4h后 ,用四唑盐比色试验法测量各孔在 490nm处的吸光度 (A值 ) ,并计算每组成纤维细胞抑制率。结果　PDT治疗后A至H组A值分别与I组A值比较 ,差异均有显著意义 (A至H组P值均为 0 0 0 0 ) ;J组与I组差异无显著意义 (P =0 2 0 3) ;A至G组的成纤维细胞抑制率分别为 93 3%、91 0 %、90 3 %、87 1%、6 6 0 %、41 6 %、12 5 %;H组的成纤维细胞抑制率为 93 0 %。结论　PDT对体外培养的人球筋膜囊成纤维细胞增殖有抑制作用 ,且抑制作用的大小随光敏剂浓度的增加而增强。